Characterization of a foxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghum bicolor

Abstract Sorghum is vulnerable to many biotic and abiotic stresses, which cause considerable yield losses globally. Efforts to genetically characterize beneficial sorghum traits, including disease resistance, plant architecture, and tolerance to abiotic stresses, are ongoing. One challenge faced by sorghum researchers is its recalcitrance to transformation, which has slowed gene validation efforts and utilization for cultivar development. Here, we characterize the use of a foxtail mosaic virus (FoMV) vector for virus‐induced gene silencing (VIGS) by targeting two previously tested marker genes: phytoene desaturase (PDS) and ubiquitin (Ub). We additionally demonstrate VIGS of a subgroup of receptor‐like cytoplasmic kinases (RLCKs) and report the role of these genes as positive regulators of early defence signalling. Silencing of subgroup 8 RLCKs also resulted in higher susceptibility to the bacterial pathogens Pseudomonas syringae pv. syringae (B728a) and Xanthomonas vasicola pv. holcicola, demonstrating the role of these genes in host defence against bacterial pathogens. Together, this work highlights the utility of FoMV‐induced gene silencing in the characterization of genes mediating defence responses in sorghum. Moreover, FoMV was able to systemically infect six diverse sorghum genotypes with high efficiency at optimal temperatures for sorghum growth and therefore could be extrapolated to study additional traits of economic importance.

A major challenge to sorghum production is its vulnerability to a wide range of bacterial, fungal, and viral pathogens (Butsenko & Reshetnikov, 2022;Little & Perumal, 2019;Sharma et al., 2015).
Disease management strategies have largely relied on leveraging natural variation in disease resistance between sorghum genotypes (Das, 2019;Mofokeng et al., 2017). Although effective at mitigating crop losses, these strategies can be time-consuming and often imprecise, resulting in unintended pleiotropic effects or yield penalties (Das, 2019). The most sustainable method to develop elite crops with durable resistance to a broad range of diseases is by identifying and introducing resistance genes using transgenic approaches.
There is therefore an urgent need to understand the genetic control of immune signalling networks in this species.
The publication and improved annotation of the sorghum reference genome (Mace et al., 2013;Mullet et al., 2014;Xin et al., 2021) have facilitated the identification of genes regulating traits of interest to sorghum growers. Numerous candidate genes for improved disease resistance have been identified, largely through differential expression analysis using susceptible and resistant genotypes (Cui et al., 2021;Lo et al., 1999;Wang et al., 2020). However, most sorghum genotypes are recalcitrant to standard transformation methods, presenting a major challenge for gene function validation. As a result, the molecular mechanisms that regulate immune responses are poorly understood compared to other staple crops.
FoMV is a member of the genus Potexvirus of plant RNA viruses and is notable for its wide host range, including 56 Poaceae species (Paulsen, 1977;Short & Davies, 1987). FoMV has a small (c.6.2 kb) monopartite genome composed of five protein-encoding open reading frames (ORFs). ORF1 encodes an RNA-dependent RNA polymerase (RdRP) that catalyses replication and transcription from two subgenomic RNAs (Rouleau et al., 1993). ORFs 2-4 encode the triple gene block (TGB) proteins involved in cell-to-cell and long-distance movement (Bruun-Rasmussen et al., 2008;Samuels et al., 2007).
ORF5 encodes the coat protein (CP), which is required for viral encapsidation and aids in viral movement (Cruz et al., 1998;Robertson et al., 2000). Previously, we and others have engineered infectious clones of FoMV capable of transiently silencing endogenous genes in Zea mays (maize), Hordeum vulgare (barley), Triticum aestivum (wheat), Setaria italica (foxtail millet), and Panicum virgatum (switchgrass) Mei et al., 2016;Tiedge et al., 2022). For the purposes of developing a system for gene function validation in sorghum, a viral vector that can infect the wide range of available genotypes is essential. We therefore infected six accessions belonging to the sorghum diversity panel (RTx430, BTx623, PI656015, PI533938, PI533936, and PI533839) (Casa et al., 2008) and assessed systemic infection 21 days postinoculation (dpi) (File S1). Reverse transcription (RT)-PCR analysis indicated that FoMV was capable of systemically infecting all six genotypes ( Figure 1a) with a 100% infection rate. Many viruses have been shown to replicate more efficiently at lower than ambient temperatures, presumably due to reduced antiviral defences (Adelman et al., 2008;Cakir & Tör, 2010;Szittya et al., 2003). FoMV appeared to efficiently replicate in systemic tissues at temperatures optimal for sorghum growth (25-28°C); this is in contrast to BMV, which replicates poorly above 22°C and most efficiently when plants are grown under low temperatures (18°C) prior to infection (Singh et al., 2018). Therefore, FoMV may be more suitable for studying gene function under agriculturally relevant conditions. RTx430 and BTx623 were selected for VIGS experiments because of their well-annotated genomes and extensive use by the sorghum research community. RTx430 was also particularly interesting to us because of the availability of optimized transformation protocols (Liu & Godwin, 2012) and thus it can be used for gene editing applications (Liu et al., 2019). To generate gene silencing constructs, approximately 300 bp fragments of PDS and Ub were selected using the Sol Genomics Network VIGS Tool (https://vigs. solge nomics.net/) and cloned in the antisense orientation at the first multiple cloning site (MCSI) of the FoMV genome ( Figure 1b, Table S1, and File S1). VIGS of these marker genes in sorghum has been demonstrated using BMV (Singh et al., 2018) and served as a basis for us to assess the relative capacity of FoMV for gene silencing. Retention of gene silencing fragments in the FoMV genome was assessed by RT-PCR analysis using primers that span the MCSI insertion site (Table S2) Figure S6). The lack of a visual phenotype associated with PDS gene silencing in sorghum is in line with previous observations using BMV (Singh et al., 2018), and could be because viral symptoms also appear as yellow spots or stripes on leaves. Alternatively, the lack of photobleaching could be due to the presence of an additional homologue of PDS (Sobic.001G480550) (Aregawi et al., 2022), which has low sequence identity to the PDS VIGS sequence and is unlikely to be silenced.
Ub has been demonstrated as an alternative visual marker for gene silencing in sorghum causing cell death (Singh et al., 2018).
Unlike PDS, FoMV-induced Ub gene silencing resulted in a strong cell death phenotype in BTx623 (Figure 1d), characterized by the development of reddish-brown lesions (Singh et al., 2018). Cell death symptoms were also observed in RTx430 plants, although this phenotype was less pronounced. Despite the clear visual phenotypes in systemic leaves, at 21 dpi a significant decrease in Ub gene expression was only observed in leaf 6 of RTx430 plants (Figure 1i,j).
Similarly, in BTx623 plants, leaves displaying a strong Ub phenotype did not reduce gene expression of Ub compared to FoMV-treated plants ( Figure 1i). We speculated that capturing Ub gene silencing could be sensitive to the timing of sample collections because strong silencing would be associated with high levels of cell death.
We therefore conducted a time-course analysis of Ub gene silencing at 14, 21, and 28 dpi. We observed silencing at 14 and 28 dpi in the newest fully expanded leaves of BTx623 plants, compared to FoMVinfected controls (Figure 1k). However, regardless of the sampling time, we were unable to verify gene silencing in RTx430 plants in this analysis (Figure 1l). Together, these results suggest that although Ub offers a clearer visual phenotype than PDS, the VIGS sequence is inherently less stable and cell death associated with gene silencing could complicate quantitative analysis using this marker gene.
We next sought to investigate the use of FoMV-mediated silencing in gene function analysis of immune genes. Plants detect pathogens using a suite of plasma membrane-localized pattern recognition receptors (PRRs) that bind conserved molecules essential to microbial life, known as microbe-associated molecular patterns (MAMPs) (Couto & Zipfel, 2016;DeFalco & Zipfel, 2021). Binding of MAMPs to cognate PRRs results in the formation of PRR/co-receptor complexes and the initiation of intracellular signalling in a process known as pattern-triggered immunity (PTI) (Chai et al., 2013;Chinchilla et al., 2006;Sun et al., 2013). PTI is the first layer of defence against pathogens and involves a complex orchestration of events culminating in broad-spectrum resistance. The second layer of defence, known as effector-triggered immunity (ETI), involves the recognition of microbial effectors that allow pathogens to evade or suppress host PTI responses by intracellular nucleotide-binding leucine-rich repeat receptors (NB-LRRs) (Adachi & Kamoun, 2022;Nguyen et al., 2021). To investigate the utility of FoMV-mediated gene silencing in gene function analysis associated with PTI, we targeted a subgroup of RLCKs with conserved roles in defence signalling in plants (Liang & Zhou, 2018;Sun & Zhang, 2020).
BTx623 was selected for immune assays because of the robust gene silencing we observed with PDS and Ub (Figure 1) and its susceptibility to bacterial and fungal pathogens (Cui et al., 2021;Patil et al., 2017). Approximately 280-300 bp sequences were selected for VIGS of individual subgroup 8 RLCKs (Table S1) Figure S7). In Arabidopsis BIK1 and PBL1 display partial redundancy in PTI signalling ; we therefore additionally coinfected plants with FoMV::RLCK1, FoMV::RLCK2, and FoMV::RLCK3 gene silencing constructs. RT-PCR analysis, using primers designed F I G U R E 2 FoMV-induced gene silencing of subgroup 8 receptor-like cytoplasmic kinases (RLCKs) in BTx623 sorghum and associated immune-elicited oxidative species production. (a) Phylogenetic tree of subgroup 8 RLCKs from Arabidopsis, rice, and sorghum generated through Clustal Multiple Sequence Alignment (https://www.ebi.ac.uk/Tools/ msa/clust alw2/) and reconstructed using the Interactive Tree of Life (iToL) web tool (Ciccarelli et al., 2006) to differentiate individual VIGS inserts, indicated that five of six plants stably expressed all three VIGS sequences ( Figure S8). RLCK1, RLCK2, and RLCK3 gene expression was substantially reduced at 21 dpi with individual gene silencing constructs compared to FoMV empty vector (FoMV::00)-treated plants (Figure 2b). Notably, we did not observe any change in RLCK gene expression in FoMV::00 infected plants compared to mock-treated controls ( Figure S9). Given the close sequence homology of these genes (Table S3), we observed some cross-silencing using FoMV::RLCK2 and FoMV::RLCK3 constructs (Figure 2b), precluding us from investigating the function of these genes individually. Cross-silencing of closely related genes is a limitation of any VIGS system, and it highlights the need to use additional approaches for further validation. We observed the most robust gene silencing of individual RLCK genes when all three FoMV gene silencing vectors were coinfected, which may be associated with silencing of individual RLCKs by multiple VIGS sequences or when infected with FoMV::RLCK3 (Figure 2b).
One of the earliest responses to MAMP detection is the activation of transmembrane RBOH proteins and the production of apoplastic reactive oxygen species (ROS), such as hydrogen peroxide (Kadota et al., 2014;Li et al., 2014). ROS serve as antimicrobial molecules as well as short and long-distance immune signals from sites of pathogen attack (Lee et al., 2020;Sun & Zhang, 2021). We therefore monitored ROS production in response to the MAMP flg22, corresponding to a 22 amino acid epitope of bacterial flagellin, known to elicit immune signalling in diverse plants including Arabidopsis, rice, soybean, and sorghum (Chinchilla et al., 2006;Cui et al., 2021;Takai et al., 2008;Wei et al., 2020). Silencing of RLCK1 alone, using the FoMV::RLCK1 silencing vector, significantly reduced flg22-elicited ROS production compared to FoMV-treated controls (Figure 2c).
The greatest reduction in ROS production was observed using the FoMV::RLCK3 construct or when all three VIGS constructs were coinfected ( Figure 2c). Some RLCKs display specificity for PRR complex activation (Rao et al., 2018), therefore we additionally monitored ROS production in response to elicitation with chitin, a component of fungal cell walls and a known MAMP in sorghum (Cui et al., 2021;Nida et al., 2021;Samira et al., 2020). Chitin elicited a more robust ROS burst than flg22 (Figure 2c,d), in line with previous observations in this species (Cui et al., 2021). Silencing of RLCK1 alone resulted in a significant reduction in chitin-elicited ROS production ( Figure 2d). bacterial diseases has received little attention in sorghum and very few mediators of bacterial defence have been identified. To determine if our VIGS system would allow us to discriminate between disease resistance associated with PTI, infection assays were conducted using two of these pathogens (File S1). Plants coinfected with all three RLCK gene silencing vectors were used for these assays because they displayed the most robust gene silencing of individual genes. RLCKsilenced plants were spray-inoculated with P. syringae (B728a) 21 days after viral inoculations. Disease severity was determined by bacterial counts (colony-forming units; cfu/cm 2 ) at 3 dpi and through visual assessment of disease symptoms at 7 dpi. RLCK-silenced plants were more susceptible to P. syringae with a roughly 1.5 log-fold increase in bacterial proliferation (Figure 3a). No differences in bacterial proliferation were observed between mock-and FoMV::00-treated plants.
Moreover, visual symptoms associated with bacterial spot disease, characterized by the appearance of irregular shaped tan lesions with red borders, were noticeably larger and more numerous on RLCK- The cultivation of sorghum as a global crop has been rapidly expanding, with more than 55 million tonnes of sorghum harvested in 2021 (Shahbandeh, 2022). Anthropogenic climate change is likely to place unforeseen pressures on sorghum production. Changes in temperature and relative humidity could affect susceptibility to plant pathogens that are not presently considered an urgent concern. It is therefore crucial to understand the genetics that underlie responses to both biotic and abiotic stresses. The successful use of FoMV-induced gene silencing for functional validation of RLCKs in this study suggests that its mild infection symptoms and ability to replicate efficiently at physiologically relevant temperatures makes this system a reliable method for validating the function of immune signalling proteins. Moreover, FoMV can silence endogenous gene expression in six distinct sorghum genotypes with high efficiency, highlighting its potential use in gene function validation in other sorghum varieties. Future studies could also utilize FoMV-mediated overexpression (Bouton et al., 2018;Mei et al., 2019) to complement functional analysis by VIGS. Together, this work provides an efficient alternative to transgenic approaches to study gene function in sorghum, overcoming the current challenges presented by its recalcitrance to transformation. State University) who provided P. syringae and X. vasicola strains, respectively. We also appreciate Joshua Kemp (Iowa State University) for providing sorghum seeds for experiments and all the technical support that we received during this study. Iowa State University is located on the ancestral territory of the Baxoje, or Ioway Nation; we are grateful to live, work, and play on these lands.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are presented in the figures and supporting materials.